Conventional characterization of a protein with enzymatic activity is expressed in terms of product formed per mass of protein present per unit time. This is referred to as the specific activity of the protein. For example, a protein that hydrolyzes a substrate to form products X and Y may be described as having a specific activity of moles of X or Y formed per gram of protein per minute
This specific activity determination requires two separate laboratory procedures 1) a measurement of the amount of product formed (moles) in a certain amount of time by a sample of the protein, and 2) an assay to determine the protein mass (gms) present in the sample. Normally the protein mass is measured in a colorimetric assay such as a Lowry protein assay or a BCA assay. The mass determination requires that a portion of the protein be sacrificed so that it may be reacted with the Lowry reagents.
The conventional specific activity characterization is limited to proteins that have bond breaking and/or making activity such as lipases, esterases, oxidases and others. The bond breaking and/or making activity is defined herein for ease of explanation of the invention as activity that makes and/or breaks chemical bonds.
For example, cholesterol ester transfer protein (CETP) is a plasma protein that shuttles lipids among lipoproteins which includes high density lipoprotein (HDL), low density lipoprotein (LDL) intermediate density lipoprotein (IDL), very low density lipoprotein (VLDL) and chylomicrons. If the plasma CETP activity is measured in a group of patients, the measurement must take into account the concentration of lipoprotein particles present in each sample. This is conventionally achieved by adding an excess of VLDL or LDL to the test sample to normalize the acceptor concentration among each sample. The conventional CETP assay would include a volume of a patient's plasma combined with a CETP compatible cholesteryl ester (CE) donor particle. The CE is labeled so that the CE mass may be quantitated after the protein shuttles the CE from donor to acceptor. A suitable acceptor is added to the plasma and donor mixture in a buffer to replicate physiological conditions. The donor, acceptor, plasma and buffer mixture is incubated. After incubation, the assay is analyzed to determine the amount of labeled CE transferred from donor to acceptor.
The added acceptor is in excess and compensates for the differences in lipoprotein particle numbers associated with each patient's plasma lipoprotein profile. If the acceptor were not added, the endogenous lipoproteins would accept the transferred CE and results of the test would vary based not only upon activity of the protein but also according to teach patient's lipoprotein profile.